The molecular basis of nervous system development remains a central question in developmental biology. Identifying the mechanisms and different pathways that orchestrate pluripotency or multipotency of stem cells, as well as the molecular signaling that coordinates lineage specification, is critical for understanding not only neural development but also neurological disorders and cancers. The RE1 silencing transcription factor, REST, and its corepressor CoREST are potential candidates for playing key roles in maintaining stem cell identity and subsequent development of the nervous system;REST/CoREST regulates a large network of genes involved in acquisition of neural fate including neuronal, proneural, and brain specific microRNA genes. In stem cells, REST/CoREST orchestrates a repressive chromatin state that is none-the-less poised for subsequent expression. REST is highly expressed in embryonic stem (ES) cells but present only in low levels in neural stem/progenitor cells. Upon differentiation, REST remains present in glia but absent in neurons, allowing selective expression of neuronal genes in neurons. The aims of the proposed research seek to systematically dissect the functional roles of REST and CoREST throughout neural development, using gain-, and loss-of-function approaches. In Aim 1, we will rigorously analyze the roles of REST/CoREST in maintaining ES cell identity, by examining the effects of loss of REST/CoREST on ES cells pluripotency and on the repressive state of neuronal gene chromatin. This will be accomplished using pluripotency tests, chromatin immunoprecipitation, microarray as well as functional analyses of aberrantly expressed key regulatory genes that are direct targets of REST/CoREST. In Aim 2, we will analyze the roles of REST/CoREST in maintaining neural stem/progenitor cell identity and in differentiation of neurogenic and gliogenic progenitors into neurons and glia. This will be accomplished by establishing enriched cortical progenitor cultures from two different embryonic stages and interfering with REST/CoREST function. Aberrantly expressed REST/CoREST target genes involved in lineage determination will be analyzed as in Aim 1. As a complementary approach to the studies proposed in Aim 2, in Aim 3, we will determine the roles of REST and CoREST in vivo during neocortical development, using in utero electroporation to interfere with REST/CoREST function. As in Aim 2, these studies will be performed at two embryonic stages when neurogenesis and gliogenesis take place. PUBLIC HEALTH RELEVANCE: REST is a key regulator of a large network of genes involved in acquisition of neuronal fate during nervous system development. Dysregulation of REST has been recently implicated in cancers in the nervous system (medulloblastoma) and outside the nervous system (epithelial cancers), as well as in neuronal death as a consequence of ischemic insults. The corepressor CoREST is an essential partner of REST, mediating differential epigenetic mechanisms to suppress REST target genes in and outside the nervous system. Like REST, CoREST has been implicated in cancer, as well as in the Notch signaling pathway, which plays critical roles in the genesis of Alzheimer disease. Thus, the proposed studies are fundamental for understanding the roles of REST and CoREST in nervous system development, as well as in neurological disorders and cancer.